1. Field
The present application relates to compositions and methods for assessing β cell loss by quantitating extrapancreatic demethylated β cell derived DNA with methylation status-specific oligonucleotide probes that target Polymerase Chain Reaction (PCR)-amplified DNA sequences of genes that are uniquely expressed by β cells.
The loss of insulin producing β cells results in glucose intolerance and the development of Type 1(T1D) and Type 2 (T2D) diabetes. Eitan Akirav, Jake A. Kushner and Kevan C. Herold, “β-Cell Mass and Type 1 Diabetes Going, Going, Gone?”, doi: 10.2337/db07-1817 Diabetes November 2008 vol. 57 no. 11 2883-2888. Currently, evaluation of β cell mass is carried out by measuring β cell products such as c-peptide. While useful, these measures do not provide real time information about active β cell loss.
There has thus been a long-felt need for a method capable of accurately evaluating β cell death so as to improve disease diagnosis, allow for disease staging, and provide a better evaluation of clinical treatment efficacy. There is further a great need in the art for compositions and methods for non-invasively monitoring β cell destruction in individuals having, or at risk of developing, diabetes mellitus (“diabetes”), including Type-1 and Type-2 Diabetes (T1D and T2D, respectively), as well as gestational diabetes.
Epigenetic modifications of DNA control cell-type specific gene expression. DNA methylation is one example of an epigenetic modification that affects gene. Methylation of DNA occurs at CpG dinucleotide sites, and this modification maintains a transcriptionally repressive chromatin configuration (Miranda et al., 2007, J. Cell Physiol. 213:384-390). Conversely, demethylation of CpG dinucleotide sites allows a transcriptionally permissive configuration (Id). Beta cells express insulin, and thus, maintain a transcriptionally-permissive hypomethylated regulatory region for the insulin gene (INS). Indeed, Genomic DNA sequences near the insulin gene are methylated in non-β cell cell types. Ley, Timothy J., et al. “DNA methylation and regulation of the human beta-globin-like genes in mouse erythroleukemia cells containing human chromosome 11.” Proceedings of the National Academy of Sciences 81.21 (1984): 6618-6622.) Therefore, the presence of hypomethylated insulin gene DNA outside of the pancreas of a subject correlated with the release of hypomethylated insulin gene DNA from dead and dying (e.g., apoptotic) β cells. Id. and Kuroda A, Rauch T A, Todorov I, Ku H T, Al-Abdullah I H, et al. (2009) Insulin Gene Expression Is Regulated by DNA Methylation. PLoS ONE 4(9): e6953. doi:10.1371/journal.pone.0006953.